PROJECT SUMMARY Poxviruses can cause deadly human and animal diseases, such as smallpox and monkeypox, and have the potential to be used as biological weapons. They are also extensively used as expression vectors for vaccine development. There is a lack of FDA-approved drugs for poxvirus-infection treatment. The development of novel strategies to prevent and treat poxvirus infections will allow for responsive treatment and management of current and re-emergent threatens posed by poxvirus infections in human and animals. Our goal in this project is to discover host cell components and mechanisms that are essential for modulating viral replication and develop strategies to disrupt these processes using a combination of genomic and chemical approaches in our studies. For our genomic approach, we will monitor global protein translation potential change in poxvirus- infected cells to identify potential cellular functions important for poxvirus replication by simultaneously and quantitatively deep sequencing total mRNAs and ribosome-associated mRNAs. We will also use a chemical approach to screen for compounds that can inhibit poxvirus replication and determine their effects on the identified host cell functions through the genomic approach. The approach may be applied to other medically relevant viruses. We will use vaccinia virus, the prototype member of poxvirus family in this study. In Specific Aim 1, we will identify potential cellular components and pathways that modulate vaccinia virus replication. In Specific Aim 2, we will discover novel chemical inhibitors of vaccinia virus replication and examine their effects on the cellular pathways and functions identified through relative translation efficiency analysis. A primary outcome of these efforts is to obtain observations and data that strongly support more mechanistic, hypothesis driven proposal still centered on the primary hypothesis that specific cellular functions modulate viral replication and these are preferentially translated during global cellular shutoff. With the study of the host translation responses to poxvirus infection and the cellular functions in poxvirus replication, we will also gain insights into the dynamic poxvirus-host interactions, which in turn, can lead to the development of preventive and therapeutic strategies for viral infections and the design of better poxvirus-based vaccines and delivery vectors.